Selenophosphate synthetase (SPS) converts ATP and selenium to selenophosphate, the selenium donor for selenoenzyme synthesis in mammals and several bacterial species. Dr. Matt Wolfe discovered that the purified E. coli enzyme contains a previously undetected bound chromophore that absorbs in the UV. This chromophore has been detected as a somewhat unstable derivative bound to one of the peptides produced by proteolytic cleavage of the protein. Studies to determine the identity of this chromophore and its possible role in the mechanism of the catalytic reaction are in progress. Rut Wattanasak, under the direction of Dr. Wolfe, has succeeded in crystallizing a mutant form of SPS. This is particularly impressive because all earlier attempts by many investigators to crystallize SPS had failed. A form of the SPS protein in which selenomethionine was substituted for methionine residues was produced and crystals of this derivative will be subjected to X-ray analysis. The selenomethionine serves as a reference point for calculation of distances to other residues in the crystal. Some needed information concerning location and amino acid composition of the active site of SPS may result from these approaches. In the usual in vitro assay for SPS activity, selenide (mM highly toxic levels) is added as the selenium substrate. As an alternative it was shown that certain selenium-binding proteins could be used to deliver an elementary form of selenium to SPS in vitro. Based on Lacourciere?s identification of glyceraldehyde-3-phosphate dehydrogenase (GADPH), a well-known glycolytic enzyme, as one of the E. coli proteins that bound selenium in vivo, experiments were designed to use GADPH as a potential selenium delivery protein in vitro. Using purified homo-tetrameric enzyme from human erythrocytes, Dr. Yuki Ogasawara showed that GADPH could bind selenium in vitro and serve as an effective selenium delivery protein. Incubation with selenodiglutathione (GSSeSG) converted the GADPH to a derivative containing 1 equivalent of bound selenium per monomer, presumably on the reactive low pKa cysteine present in each subunit. This selenium was transferred to SPS in the in vitro assay with ATP and converted to selenophosphate. The gene encoding a selenium-binding protein previously isolated from Methanococcus vannielii was cloned and expressed in E. coli by Dr. William Self. The recombinant protein consists of 8.8 kDa subunits and is very hydrophobic. Physical chemical studies carried out by Dr. Kemberly Patteson served to explain the ability of the isolated protein to retain bound selenium. A single cysteine residue located in each subunit is required for selenium binding. This cysteine residue is completely buried both in oxidized and reduced forms of the protein and it was necessary to partially unfold the protein to detect its chemical reactivity. Experiments in progress by Dr. Michelle Galloway indicate that an affinity matrix containing covalently bound selenophosphate synthetase that she has prepared has the potential to react selectively with a selenium binding protein. In preliminary tests using GADPH, this protein bound to the affinity matrix and could be eluted with KCl containing DTT. This approach may prove useful for detection and isolation of potential selenium delivery proteins from various sources.